Project Summary The ribonucleoprotein complex, telomerase, counteracts loss of terminal sequences during DNA replication. Inappropriate activation of telomerase facilitates immortal growth of human tumor cells, raising interest in this enzyme as a chemotherapeutic target. In Saccharomyces cerevisiae, telomerase contains an RNA template (TLC1 RNA), a reverse transcriptase (Est2p), and at least two additional protein components, Est1p and Est3p. Est2p is constitutively telomere-bound, while Est1p associates with telomeres in late S phase, coincident with telomere lengthening. Previous work from the P.I.'s laboratory demonstrated that the regulated degradation of Est1p during G1 phase prevents assembly of the telomerase complex. Stabilization of Est1p during G1 phase by proteasome inhibition promotes association of both Est1p and Est3p with Est2p, uncovering a novel role of Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, enzyme assembled during G1 does not lengthen telomeres, suggesting that additional regulatory events must occur to activate the enzyme as cells transit S phase. The experiments proposed here are designed to address critical questions regarding the mechanism of telomerase assembly and activation in the yeast cell cycle. Because the Est2p and Est1p subunits are conserved in human telomerase, these experiments will facilitate the long-term goal of identifying regulatory events that may serve as targets of telomerase inactivation in human tumor cells. Aims of this proposal are to (1) determine the regulatory pathway(s) that impinge upon Est1p degradation; (2) examine the mechanism through which Est1p facilitates complex assembly; and (3) characterize the kinetics of telomerase assembly and activation in cells transiting a synchronous cell cycle. These experiments will provide the first detailed description of protein interactions that occur in the telomerase complex as cells undergo DNA replication. Novel tools will be developed to address the mechanism(s) that restrict telomerase activity during G1 phase and to probe the functional relevance of this regulation. In addition, cis-acting mutations in telomerase components and/or defects in trans-acting regulatory pathways that perturb telomerase assembly will be identified. The goals of this work are to describe the mechanisms regulating telomerase during the cell cycle and to elucidate the consequences of these events for telomerase activity and telomere maintenance. General Relevance In most human cells, repeated rounds of cell division result in chromosome shortening, a phenomenon that restricts the number of times a cell can divide. Tumor cells often circumvent this limitation by activating an enzyme called telomerase that is capable of replacing DNA sequences lost during replication. By examining the mechanisms that regulate telomerase activity, we hope to identify novel targets for anti-tumor therapy.